Regina W. Wang

Characterization of Mice Lacking the Multidrug Resistance Protein Mrp2 (Abcc2)

The multidrug resistance protein Mrp2 is an ATP-binding cassette (ABC) transporter mainly expressed in liver, kidney, and intestine. One of the physiological roles of Mrp2 is to transport bilirubin glucuronides from the liver into the bile. Current in vivo models to study Mrp2 are the transporter-deficient and Eisai hyperbilirubinemic rat strains. Previous reports showed hyperbilirubinemia and induction of Mrp3 in the hepatocyte sinusoidal membrane in the mutant rats. In addition, differences in liver cytochrome P450 and UGT1a levels between wild-type and mutant rats were detected. To study whether these compensatory mechanisms were specific to rats, we characterized Mrp2–/– mice. Functional absence of Mrp2 in the knockout mice was demonstrated by showing increased levels of bilirubin and bilirubin glucuronides in serum and urine, a reduction in biliary excretion of bilirubin glucuronides and total glutathione, and a reduction in the biliary excretion of the Mrp2 substrate dibromosulfophthalein. To identify possible compensatory mechanisms in Mrp2–/– mice, the expression levels of 98 phase I, phase II, and transporter genes were compared in liver, kidney, and intestine of male and female Mrp2–/– and control mice. Unlike in Mrp2 mutant rats, no induction of Mrp3 in Mrp2–/– mice was detected. However, Mrp4 mRNA and protein in liver and kidney were increased ∼6- and 2-fold, respectively. Phenotypic analysis of major cytochrome P450-mediated activities in liver microsomes did not show differences between wild-type and Mrp2–/– mice. In conclusion, Mrp2–/– mice are a new valuable tool to study the role of Mrp2 in drug disposition.

CYTOCHROME P450 3A4 IS THE MAJOR ENZYME INVOLVED IN THE METABOLISM OF THE SUBSTANCE P RECEPTOR ANTAGONIST APREPITANT

The contribution of human cytochrome P450 (P450) isoforms to the metabolism of aprepitant in humans was investigated using recombinant P450s and inhibition studies. In addition, aprepitant was evaluated as an inhibitor of human P450s. Metabolism of aprepitant by microsomes prepared from baculovirus-expressed human P450s was observed only when CYP1A2, CYP2C19, or CYP3A4 was present in the expression system. Incubation with CYP1A2 and CYP2C19 yielded only products of O-dealkylation, whereas CYP3A4 catalyzed both N- and O-dealkylation reactions. The metabolism of aprepitant by human liver microsomes was inhibited completely by ketoconazole or troleandomycin. No inhibition was observed with other P450 isoform-selective inhibitors. Aprepitant was evaluated also as a P450 inhibitor in human liver microsomes. No significant inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP2E1 was observed in experiments with isoform-specific substrates (IC50 > 70 μM). Aprepitant was a moderate inhibitor of CYP3A4, with Ki values of ∼10 μM for the 1′- and 4-hydroxylation of midazolam, and the N-demethylation of diltiazem, respectively. Aprepitant was a very weak inhibitor of CYP2C9 and CYP2C19, with Ki values of 108 and 66 μM for the 7-hydroxylation of warfarin and the 4′-hydroxylation of S-mephenytoin, respectively. Collectively, these results indicated that aprepitant is both a substrate and a moderate inhibitor of CYP3A4.

Evidence for the bioactivation of zomepirac and tolmetin by an oxidative pathway. Identification of glutathione adducts in vitro in human liver microsomes and in vivo in rats

Although zomepirac (ZP) and tolmetin (TM) induce anaphylactic reactions and form reactive acyl glucuronides, a direct link between the two events remains obscure. We report herein that, in addition to acyl glucuronidation, both drugs are subject to oxidative bioactivation. Following incubations of ZP with human liver microsomes fortified with NADPH and glutathione (GSH), a metabolite with an MH+ ion at m/z 597 was detected by LC/MS/MS. On the basis of collision-induced dissociation and NMR evidence, the structure of this metabolite was determined to be 5-[4′-chlorobenzoyl]-1,4-dimethyl-3-glutathionylpyrrole-2-acetic acid (ZP-SG), suggesting that the pyrrole moiety of ZP had undergone oxidation to an epoxide intermediate, followed by addition of GSH and loss of the elements of H2O to yield the observed conjugate. The oxidative bioactivation of ZP most likely is catalyzed by cytochrome P450 (P450) 3A4, since the formation of ZP-SG was reduced to ∼10% of control values following pretreatment of human liver microsomes with ketoconazole or with an inhibitory anti-P450 3A4 IgG. A similar GSH adduct, namely 5-[4′-methylbenzoyl]-1-methyl-3-glutathionylpyrrole-2-acetic acid (TM-SG), was identified when TM was incubated with human liver microsomal preparations. The relevance of these in vitro findings to the in vivo situation was established through the detection of the same thiol adducts in rats treated with ZP and TM, respectively. Taken together, these data suggest that, in addition to the formation of acyl glucuronides, oxidative metabolism of ZP and TM affords reactive species that may haptenize proteins and thereby contribute to the drug-mediated anaphylactic reactions.

Assessment of the CYP3A‐Mediated Drug Interaction Potential of Anacetrapib, a Potent Cholesteryl Ester Transfer Protein (CETP) Inhibitor, in Healthy Volunteers

In this study, midazolam was used as a probe‐sensitive CYP3A substrate to investigate the effect of anacetrapib on CYP3A activity, and ketoconazole was used as a probe‐inhibitor to investigate the effect of potent CYP3A inhibition on the pharmacokinetics of anacetrapib, a novel cholesteryl ester transfer protein inhibitor in development for the treatment of dyslipidemia. Two partially blinded, randomized, 2‐period, fixed‐sequence studies were performed. Safety, tolerability, and midazolam and anacetrapib plasma concentrations were assessed. All treatments were generally well tolerated. The geometric mean ratios (90% confidence interval) of midazolam with anacetrapib/midazolam alone for AUC0‐∞ and Cmax were 1.04 (0.94, 1.14) and 1.15 (0.97, 1.37), respectively. Exposure to anacetrapib was increased by ketoconazole—specifically, the geometric mean ratios (90% confidence interval) of anacetrapib with ketoconazole/anacetrapib alone for AUC0‐∞ and Cmax were 4.58 (3.68, 5.71) and 2.37 (2.02, 2.78), respectively. The study showed that anacetrapib does not inhibit or induce CYP3A activity. Furthermore, anacetrapib appears to be a moderately sensitive substrate of CYP3A.

Utility of Recombinant Cytochrome P450 Enzymes: A Drug Metabolism Perspective

An important role of human cytochrome P450s (P450s) has been well recognized in the area of drug metabolism and pharmacokinetics. It has become possible in recent years to express catalytically active forms of these enzymes in various host systems. The resulting recombinant human P450s are either purified for studies of protein structure and the mechanism of catalysis or isolated in microsomal forms to serve the purposes of P450 phenotyping, metabolic stability screening and inhibitory potential evaluation. Intact mammalian cells expressing human enzymes may also be used to test the mutagenic and toxicity potential of drug candidates. The issue remains, however, that the data derived from recombinant P450s are not always consistent with those generated from human tissue preparations. The aim of this communication is to discuss applications of recombinant P450s in the drug discovery and development setting, with an emphasis on comparison of recombinant and human liver microsomal systems.

Rat liver DT-diaphorase: Regulation of functional mRNA levels by 3-methylcholanthrene, trans-stilbene oxide, and phenobarbital

Total liver poly(A+)-RNA isolated from untreated, and 3-methylcholanthrene-, trans-stilbene oxide-, and phenobarbital-treated rats has been translated in the rabbit reticulocyte lysate system in order to determine the effect of these xenobiotics on the level of translationally active DT-diaphorase mRNA. The in vitro translation systems were subjected to immunoprecipitation with rabbit IgG raised against purified DT-diaphorase and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The identity of the radiolabeled, immunoprecipitated product as DT-diaphorase was confirmed by limited peptide mapping using Staphylococcus aureus V-8 protease. These quantitation results demonstrate that 3-methylcholanthrene leads to an eight-fold elevation in functional DT-diaphorase mRNA at 8 h after a single administration of 3-methylcholanthrene; whereas, trans-stilbene oxide and phenobarbital produced only a modest elevation, two- to three-fold, in the functional DT-diaphorase mRNA level. These data indicate that the increase in the level of DT-diaphorase after 3-methylcholanthrene administration noted previously [B. Höjeberg, K. Blomberg, S. Stenberg, and C. Lind (1981) Arch. Biochem. Biophys. 207, 205-216] can be totally accounted for by an elevation in the mRNA level specific for this protein.

Site-directed mutagenesis of glutathione S-transferase YaYa: Nonessential role of histidine in catalysis

A cDNA encoding a rat liver glutathione S-transferase Ya subunit has been expressed in Escherichia coli and the expressed enzyme purified to homogeneity. In order to examine the catalytic role of histidine in the glutathione S-transferase Ya homodimer, site-directed mutagenesis was used to replace all three histidine residues (at positions 8, 143, and 159) by other amino acid residues. The replacement of histidine 8 or histidine 143 with valine did not affect the 1-chloro-2,4-dinitrobenzene-conjugating activity nor the isomerase activity. However, the replacement of histidine with valine at position 159 produced the mutant GST which exhibited only partial activity. A greater decrease in catalytic activity was observed by histidine----tyrosine or histidine----lysine replacement at position 159. On the other hand, the histidine 159----asparagine mutant retained full catalytic activity. Our results indicate that histidine residues in the Ya homodimer are not essential for catalytic activity. However, histidine 159 might be critical in maintaining the proper conformation of this enzyme since replacement of this amino acid by either lysine or tyrosine did result in significant loss of enzymatic activity.

Assessing and minimizing time-dependent inhibition of cytochrome P450 3A in drug discovery: A case study with melanocortin-4 receptor agonists

1-[(2R)-2-({[(1S,2S)-1-amino-1,2,3,4-tetrahydronaphthalen-2-yl]carbonyl}amino)-3-(4-chlorophenyl)propanoyl]-N-(tert-butyl)-4-cyclohexylpiperidine-4-carboxamide (1) is a potent melanocortin-4 receptor agonist that exhibited time-dependent inhibition of cytochrome P450 (P450) 3A in incubations with human liver microsomes. In incubations fortified with potassium cyanide, a cyano adduct was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as a cyanonitrosotetrahydronaphthalenyl derivative. The detection of this adduct suggested that a nitroso species was involved in the formation of a metabolite intermediate (MI) complex that led to the observed P450 inactivation. Further evidence supporting this hypothesis derived from incubations of 1 with recombinant P450 3A4, which exhibited a λmax at approximately 450 nm. The species responsible for this absorbance required the presence of β-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), increased with increasing incubation time and decreased following the addition of potassium ferricyanide to the incubation mixture, suggestive of an MI complex. Similar results were obtained with rat liver microsomes and with recombinant P450 3A1. When rats were dosed with indinavir as a P450 3A probe substrate, plasma exposure to indinavir increased three-fold following pretreatment with 1, consistent with drug–drug interaction projections based on the kinact and KI parameters for 1 in rat liver microsomes. A similar approach was used to predict the magnitude of the corresponding drug–drug interaction potential in humans dosed with a drug metabolized predominantly by P450 3A, and the forecast area under the curve (AUC) increase ranged from four- to ten-fold. These data prompted a decision to terminate further evaluation of 1 as a development candidate, and led to the synthesis of the methyl analogue 2. Methyl substitution α to the amino group in 2 was designed to reduce the propensity for formation of a nitroso intermediate and, indeed, 2 failed to exhibit time-dependent inhibition of P450 3A in human liver microsomal incubations. This case study highlights the importance of mechanistic studies in support of drug-discovery and decision-making processes.

Multiple Ya subunits of glutathione S-transferase detected by monoclonal antibodies

Monoclonal antibodies to ligandin (YaYa) and glutathione (GSH) S-transferase B (YaYc) were produced by hybridomas derived from the fusion of mouse myeloma cells and spleen cells of mice immunized with the YaYa or YaYc proteins, respectively. Enzyme-linked immunosorbent assay was used to screen for antibody-producing clones. Immunoblotting of the subunits of transferase B, ligandin, and another GSH S-transferase containing Yb subunits showed that the monoclonal antibodies produced by two anti-YaYa subclones recognized the Ya subunits of both ligandin and transferase B, but they did not bind Yc or Yb subunits. It was also revealed that antibodies produced by several anti-YaYc subclones recognized the Yc subunit, but not the Ya subunit of the antigen which was used for the immunization of the mice. However, these monoclonal antibodies did bind the Ya subunit of ligandin. These results indicate that the Ya subunits of GSH S-transferase B and of ligandin do share at least one common determinant. However, these two Ya subunits are structurally distinct as evidenced by their differences in binding by monoclonal anti-YaYc antibodies.

Drug residue formation from ronidazole, a 5-nitroimidazole. VII. Comparison of protein-bound products formed in vitro and in vivo

In vivo experiments were conducted with ronidazole radiolabelled in the 2-14CH2-, 4,5-14C-, N-14CH3- and 4-3H-positions. The hepatic protein-bound residues, assessed by the radioactivity of exhaustively washed protein samples, were independent of the radiolabel position and occurred with 4-3H loss (greater than 80%) in excellent agreement to previous results obtained in vitro with anaerobic incubations of liver microsomes (Miwa et al., Chem. Biol. Interact., 41 (1982) 297). HPLC analysis of acid hydrolyzed in vivo protein-bound residues, obtained from [2-14CH2] ronidazole, produced a radiochromatographic profile which was virtually identical to that obtained from a similarly treated in vitro sample. Moreover, almost quantitative (76-96%) liberation of radiolabelled methylamine was obtained from hydrolysates of in vivo and in vitro residue samples formed from [N-14CH3] ronidazole. With 4,5-ring labeled ronidazole the distribution of total radioactivity of the protein hydrolysate on cation exchange resin and the fraction of the residue recovered as oxalic acid were nearly identical for the in vivo and in vitro products. We interpret these data to indicate that ronidazole alkylates proteins with retention of most of the carbon framework of the molecule, in vivo. It is also concluded that the in vitro model, previously used to examine the mechanism of protein alkylation, accurately reflects the salient process initially occurring in the intact animal during the formation of protein-bound residues of this drug.